Bioconjugated chelates based on (methylpyridinyl)tacn: synthesis, 64Cu labeling and in vitro evaluation for prostate cancer targeting

International audienceAbstract Three new bifunctional copper chelators based on the 1,4,7-triazacyclononane (tacn) platform have been synthesized and conjugated to peptide. The first one is constituted of the tacn with two methylpyridinyl and one methylthiazolyl carboxylic acid pendant arms, while, in the second and third ones, the macrocycle is functionalized by three methylpyridinyl groups, with an additional hexynoic acid chain on a carbon of one or two pyridine rings. These three bifunctional chelators have been conjugated to the antagonist JMV594 peptide for targeting the gastrin releasing peptide receptor (GRP-r), which is overexpressed in prostate cancer. The resulting monomeric bioconjugates have shown their efficiency to be radiolabeled with β+ emitter 64Cu, and the hydrophilicity and PC-3 cell internalisation properties of these radiolabeled conjugates have been studied. PC-3 cell binding affinity of mono- and dimeric metal-free and natCu metallated conjugates have been evaluated by IC50 measurements. The results demonstrate the potential of these methylpyridinyl tacn derivatives for radiopharmaceutical applications

New Triazacycloalkane Derivatives as Cytotoxic Agents for CLL Treatment: From Proof of Concept to the Targeting Biomolecule

International audienceThe 1,4,7-tris-(2-pyridinylmethyl)-1,4,7-triazacyclononane ligand (no3py) and its bifunctional analogue no3pyCOOK were synthesized to investigate their action toward zinc(II) depletion related to the apoptosis phenomenon in chronic lymphocytic leukemia (CLL) cells. no3py was used as the “free” ligand, while its “graftable” derivative was conjugated on a newly synthesized bifunctional sialoglycan, 6′-SL-NH2, selected to specifically bind CD22 biomarker expressed on the B-CLL cell surface. Both compounds were produced with good yields thanks to a Sonogashira coupling reaction and an orthoester function, respectively, for the chelator and the targeting moiety. The newly reported bioconjugate 6′-SL-no3py was then obtained through a peptidic coupling reaction. Biological in vitro studies of no3py and 6′-SL-no3py consisting of real-time detection of cell health (cytotoxicity and proliferation) and caspases 3/7 activation (crucial enzymes whose activation triggers cell death signaling pathways) have been investigated. First, Ramos, Daudi, and Raji B-cell lines, which present different sensitivity to zinc(II) content variation, were incubated with no3py and 6′-SL-no3py. Then, a videomicroscope allowed the real-time monitoring of the morphological changes leading to cell death from the detection of the cytotoxicity, the antiproliferative effect, and the caspasic activity. In terms of mechanism, the Zn2+ chelator cytotoxic effect of no3py has been evidenced by a culture medium ion supplementation study and by the decrease of intracellular fluorescence of Zn-specific fluorophore zinquin in the presence of no3py and 6′-SL-no3py chelators. Finally, flow cytometry analysis with classical Annexin V staining was conducted to detect no3py- and 6′-SL-no3py-induced apoptotic cell death in B-CLL cells. Time-course analysis, using the Incucyte Live-Cell Analysis System, demonstrated that no3py induced cell death in a time- and dose-dependent manner with variability across cell lines. 6′-SL-no3py exhibited the same dose-dependent trend as no3py, showing the efficiency of the targeting moiety. In both cases, the chelators depicted proliferation curves that were inversely correlated with kinetic death. Morphological changes specific to apoptosis and caspase 3/7 activation were observed for the three cell lines treated with no3py and 6′-SL-no3py, highlighting their role as apoptotic agents. A higher concentration of 6′-SL-no3py is needed to reach 50% of the B-CLL mortality, confirming a targeting of the chelator to the cell membrane. Overall, our results proved that the biological properties of the triazamacrocyclic chelator still remain even after addition of the targeting moiety. The free chelator as well as the bioconjugate constitute promising cytotoxic agents for CLL therapy through apoptosis induction

H-3 nota Derivatives Possessing Picolyl and Picolinate Pendants for Ga3+ Coordination and Ga-67(3+) Radiolabeling

International audienceWe synthesized, thanks to the regiospecific N-functionalizationusing an orthoamide intermediate, two 1,4,7-triazacyclononane derivativescontaining an acetate arm and either a methylpyridine or a picolinicacid group, respectively, Hnoapy and H-2 noapa, as new Ga3+ chelators for potential usein nuclear medicine. The corresponding Ga3+ complexes weresynthesized and structurally characterized in solution by H-1 and C-13 NMR. The [Ga(noapy)](2+) complex appears to exist in solution as two diasteroisomeric pairsof enantiomers, as confirmed by density functional theory (DFT) calculations,while for [Ga(noapa)](+), a single species ispresent in solution. Solid-state investigations were possible forthe [Ga(noapa)](+) complex, which crystallizedfrom water as a pair of enantiomers. The average length of the N-Gabonds of 2.090 and ANGS; is identical with that found for the [Ga(nota)] complex, showing that the presence of the picolinatearm does not hinder the coordination of the ligand to the metal ion.Protonation constants of noapy ( - ) and noapa ( 2- ) weredetermined by potentiometric titrations, providing an overall basicity n-ary sumation log K ( i ) (H) (i = 1-4) that increases in the order noapy ( - ) < noapa ( 2- ) < nota ( 3- ) with increases in the negative charge of theligand. Stability constants determined by pH-potentiometric titrationssupplemented with Ga-71 NMR data show that the stabilitiesof [Ga(noapy)](2+) and [Ga(noapa)](+) are lower compared to that of [Ga(nota)] but higher than those of other standards such as [Ga(aazta)](-). Ga-67 radiolabeling studies wereperformed in order to demonstrate the potential of these chelatorsfor Ga-67/68-based radiopharmaceuticals. The labelings ofHnoapy and H-2 noapa were nearlyidentical, outperforming H-3 nota. Stabilitystudies were conducted in phosphate-buffered saline and in the presenceof human serum transferrin, revealing no significant decomplexationof [Ga-67][Ga(noapy)](2+) and [Ga-67][Ga(noapa)](+) compared to [Ga-67][Ga(nota)]. Finally, all complexes were foundto be highly hydrophilic, with calculated log D (7.4) values of -3.42 and PLUSMN; 0.05, -3.34 and PLUSMN;0.04, and -3.00 and PLUSMN; 0.23 for Hnoapy, H-2 noapa, and H-3 nota, respectively,correlating with the charge of each complex and the electrostaticpotentials obtained with DFT. Hnoapy and H-2 noapa havebeen synthesized and studied as H-3 nota alternativesfor the complexation of Ga3+ and Ga-67/68, providingenhanced stability of the radiocomplex while maintaining good coordinationproperties